Methods for Enhanced Somatostatin Immunogenicity in the Treatment of Obesity

ABSTRACT

Compositions and methods are provided for treatment growth hormone and/or insulin-like growth factor 1 deficiency in a patient in need of such treatment. Compositions and methods include novel vaccines that provide immunogenicity for somatostatin and result in the increased release of endogenously produced growth hormone and/or insulin-like growth factor 1.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/523,952, entitled “Methods for Enhanced Somatostatin Immunogenicityin the Treatment of Obesity”, filed Jun. 24, 2009, which is a 35 U.S.C.§371 national phase application of International Patent ApplicationSerial Number PCT/US2009/048429, filed Jun. 24, 2009, entitled“Compositions and Methods for Enhanced Somatostatin Immunogenicity inthe Treatment of Growth Hormone and Insulin-Like Growth Factor OneDeficiency”, which application claims the benefit of U.S. ProvisionalPatent Application Ser. No. 61/075,656, filed Jun. 25, 2008, entitled“Compositions and Methods for Enhanced Somatostatin Immunogenicity inthe Treatment of Growth Hormone Deficiency”, and is related toInternational Patent Application Serial Number PCT/US08/68195, filed onJun. 25, 2008, entitled “Chloramphenicol Acetyl Transferase(CAT)-Defective Somatostatin Fusion Protein and Uses Thereof”, each ofwhich are incorporated herein by reference for all purposes in entirety.

Incorporated by reference herein in its entirety is the Sequence Listingentitled “2013_(—)02_(—)18_SEQ_LISTING_(—)40830_(—)0003USC1_TXT”,created Feb. 18, 2013 size of 16.0 kilobytes.

TECHNICAL FIELD

The present invention relates to treatments of patients having growthhormone (GH) and/or insulin-like growth factor 1 (IGF-1) deficiency.More particularly, the invention relates to treatments, and thecompositions and methods involved in said treatments, for patientshaving GH and/or IGF-1 deficiency through use of somatostatin-basedantigen/adjuvant vaccines.

BACKGROUND

Prevention of infectious disease using vaccines has been in practicesince the late 1700s (smallpox vaccine of 1798), including use ofvaccines for prevention of polio, hepatitis B, and influenza. Morerecently, vaccines have also been identified for use in treatment ofcancer, where the vaccine coaxes the patient's immune system intoidentifying and destroying target tumor cells, i.e., treatment of breastcancer, colon cancer, skin cancer, etc. Other new and useful targets forvaccine treatment are being developed due to the advantage of using apatient's own immune system to defeat the invading or cancerous agent.In most instances, a vaccine combines an antigen against which theimmunity is sought and an adjuvant to enhance the response to thatantigen by the recipient of the vaccine.

Growth hormone is a 191 amino acid polypeptide hormone synthesized andreleased from the anterior pituitary gland. Growth hormone is generallyconsidered an anabolic hormone, required for growth/height in children,increase in calcium retention (bone strength), promotion of lipolysis,increase in protein synthesis, promotion of gluconeogenesis in the liverand other like functions. Patients who suffer from endogenous growthhormone deficiency often have poor bone density, diminished lean bodymass, reduced energy, and other like general symptoms.

Presently, patients suffering from growth hormone deficiency are treatedwith growth hormone replacement, typically using a recombinant growthhormone expressed in genetically engineered bacteria. These treatmentprotocols are typically extremely costly (estimates range from $10,000to $30,000 a year) and rely on exogenous replacement of the hormone(daily injections are typical, often spanning 18 months to the lifetimeof the patient). As a result, novel, non-exogenous, long-acting, andless costly therapies are required for patients suffering from growthhormone deficiency.

In addition, therapies are also needed to treat patients in need ofadditional, above baseline levels, of growth hormone, for example thesepatients often require additional growth hormone for the treatment ofobesity, wound healing, burn healing, etc.

The present invention is directed toward overcoming one or more of theproblems discussed above.

SUMMARY OF THE EMBODIMENTS

Embodiments of the present invention provide compositions and methodsfor treatment of a growth hormone deficiency. For purpose of theinvention, a growth hormone deficiency is any decrease in levels ofgrowth hormone associated with a disease state or growth failure due tolack of adequate endogenous growth hormone secretions and/or levels.Growth hormone deficiency also includes situations where a normalendogenous level (for that patient) of growth hormone exists, butadditional growth hormone is believed advantageous for the treatment ofa disease or condition, for example, treatment of obesity, treatment ofwounds, and treatment of burns.

Embodiments of the present invention also provide compositions andmethods for treatment of an insulin-like growth factor 1 deficiency. Forpurpose of the invention, an insulin-like growth factor 1 deficiency isany decrease in levels of insulin-like growth factor 1 associated with adisease state or condition due to lack of adequate endogenousinsulin-like growth factor 1 secretions and/or levels. Insulin-likegrowth factor 1 deficiency also includes situations where a normalendogenous level of insulin-like growth factor 1 exists, but additionalinsulin-like growth factor 1 is believed advantageous for the treatmentof a disease or condition, for example, growth failure, types 1 and 2diabetes, and cartilage repair and/or replacement.

In one embodiment, methods are provided for treatment of a growthhormone deficiency in a patient in need thereof. Methods includeadministering an immunogenic amount of a somatostatin-based antigenvaccine of the invention to a patient and monitoring the patient'sprogress. Additional vaccinations can be administered to facilitatetreatment of the patient's growth hormone deficiency. Patients in needof such treatment include: adult and children that have: a lack ofendogenous growth hormone that results in insufficient growth,congenital heart conditions or other like cardiac diseases, obesity,patients in need of enhanced burn repair, and patients in need ofenhanced wound healing.

In another embodiment, methods are provided for treatment of aninsulin-like growth factor 1 deficiency in a patient in need thereof.Methods include administering an immunogenic amount of asomatostatin-based antigen vaccine of the invention to a patient andmonitoring the patient's progress. Additional vaccinations can beadministered to facilitate treatment of the patient's insulin-likegrowth factor 1 deficiency. Patients in need of such treatment include:infants that have retinopathy of prematurity (ROP), adults and/orchildren that are obese, adults and/or children that have type 1 or 2diabetes, adults and/or children that have Rett's syndrome, dogs and/orcats that are obese, horses that require replacement and/or repair ofcartilage, and other like treatments.

The present invention also provides novel polypeptides, and thepolynucleotides that encode them, having enhanced immunogenicity ofsomatostatin for use in treatment of a patient having a growth hormoneor insulin-like growth factor 1 deficiency. Polypeptides of theinvention include somatostatin-14 fused to an inactivatedchloramphenicol acetyl transferase protein via a functionally optimizedlinker. Polypeptides of the invention are useful in all vertebraespecies due to the highly conserved nature of somatostatin-14, stablefor long term storage, highly immunogenetic, and resistant todegradation in the patient. As such, the chimeric polypeptides of theinvention provide highly effective and low cost materials for use intreatment of growth hormone and/or insulin-like growth factor 1deficiency in all vertebrae.

The present invention also provides novel adjuvants for use in thetreatment of a patient in need of an immunogenic response, especially inrelation to a patient in need of a treatment of either a growth hormoneor insulin-like growth factor deficiency. Novel adjuvants herein arehighly effective and safe for use in vertebrates, including humans,dogs, horses, cats, and the like.

The present invention also provides novel vaccines for use in thetreatment of a patient having either a growth hormone or insulin-likegrowth factor deficiency. Vaccines are highly effective and safe for usein vertebrates, including humans, dogs, horses, cats and the like.

These and various other features and advantages of the invention will beapparent from a reading of the following detailed description and areview of the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustrative schematic of a pET30b CatSom plasmid inaccordance with embodiments of the present invention. The plasmidincludes a Kanamycin resistance marker, a Lac operator, T7 promoter, CATcoding sequence all in accordance with embodiments of the invention, alinker region in accordance with the invention herein and a somatostatinencoding region in accordance with the invention are also included.

FIG. 2 is an illustrative stained SDS-PAGE showing a 28 KD bandcorresponding to the predicted size of a codon-optimized, CAT-defectivesomatostatin polypeptide of the invention. Lane 1 is LB+IPTG, reduced,Lane 2 is LB, reduced, Lane 3 is LB+IPTG and Lane 4 is LB.

FIG. 3 is a scatter graph of percent baseline weight versus somatostatincontaining vaccinations on outbred mice.

FIG. 4 is a graph showing the intake of food per group of mice over acourse of 7 days.

FIG. 5 is a graph showing the mean body weight of treatment groups ofmice over the course of 39 days, each group tested is shown with anerror bar.

FIG. 6 is a graph showing percent of baseline body weight of treatmentgroups of mice over the course of 39 days, each group testing is shownwith an error bar.

IDENTIFICATION OF SEQUENCES AND SEQUENCE IDENTIFIERS

SEQ ID NO: 1 AGCKNFFWKTFTSC SEQ ID NO: 15GCTGGCTGCAAGAATTTCTTCTGGAAGACTTTCACATCCTGT (His192−>Gly, His193−>Gly):SEQ ID NO: 2 Atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttggtggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcag (His192−>Gly, His193−>Gly):SEQ ID NO: 3: Mekkitgyttvdisqshrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgkdvlmplaiqvggavcdgfhvgrmlnelqq (His193−>Gly) SEQ ID NO: 4Atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcag (1 His193−>Ala) SEQ ID NO: 5Atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatgctgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcag (1 His + CAT wt) SEQ ID NO: 6Atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatggtgccgtttgtgatggcttccatgtcggcagaatgcttaatgaactgcagcag (one H−>G) SEQ ID NO: 7Mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdegelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfiennmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvhgavcdgfhvgrmlnelqq (H−>A) SEQ ID NO: 8:Mekkitgyttvdisqwhrkehfeafqsvaqctunqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvhaavcddgfhvgrmlnelqq SEQ ID NO: 9tgggaactgcaccgttctggtccacgcccgcgccctcgcccacgtc cggaattcatg SEQ ID NO: 10welhrsgprprprprefm SEQ ID NO: 11 welhrsgp(rp)_(n)efm where n > 1SEQ ID NO: 12 Atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttggtggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcagtgggaactgcaccgttctggtccacgcccgcgccctcgcccacgtccggaattcatggccggctgcaagaacttcttttggaaaacctttacgagctgc SEQ ID NO: 13mekkitgyttvdisqwhrkehteafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdeshpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyyqgdkvlmplaiqvggavcdgfhgrmlnelqqwelhrsgprprprprpefmagc knffwktftsc SEQ ID NO: 14mekkitgytgtvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvhhavcdgfhvgrmlnelqqselhrsgprprprprpefm agcknffwktftsc

DETAILED DESCRIPTION

The present invention provides compositions and methods for treatment ofgrowth hormone deficiency in patients in need of such treatment. Forpurposes herein, and as described previously, a growth hormonedeficiency is any decrease in levels of growth hormone associated with adisease state or condition due to lack of adequate endogenous growthhormone secretions and/or levels in the patient. Growth hormonedeficiency also includes situations where a normal endogenous level ofgrowth hormone exists, i.e., normal for the patient, but additionalgrowth hormone is believed advantageous to the patient for the treatmentof a target disease or condition, for example, treatment of obesity,treatment of wounds, treatment of burns, etc.

The present invention also provides compositions and methods fortreating insulin-like growth factor deficiency 1 in patients in need ofsuch treatment. For purposes herein, an insulin-like growth factor 1deficiency is any deficiency in levels of insulin-like growth factorassociated with a disease state due to lack of adequate endogenousinsulin-like growth factor 1 secretions. Insulin-like growth factor 1deficiency also includes situations where a normal level of insulin-likegrowth factor exists, i.e., normal for the patient, but additionalfactor 1 is advantageous to the patient for the treatment of a diseaseor condition, for example, obesity, type 1 and 2 diabetes, and Rett'ssyndrome.

In one embodiment, novel polypeptides, and the polynucleotides thatencode them, are provided including polypeptides of somatostatin-14fused to an inactivated chloramphenicol acetyl transferase protein via afunctionally optimized linker. The chimeric polypeptides of theinvention provide highly effective and low cost materials for use intreatment of growth hormone and/or insulin-like growth factor 1deficiency.

In another embodiment, novel adjuvant compositions are provided for usein treatment of patient's having growth hormone and/or insulin-likegrowth factor 1 deficiency. In one particular embodiment,somatostatin-based antigen can be combined with novel adjuvants and usedin the treatment of growth hormone or insulin-like growth factor 1deficiency related disease states or conditions, e.g., growth deficiencyin children, growth deficiency in adults, lack of adequate endogenousgrowth hormone secretions, healing of burns, obesity, cardiac disease,etc. Adjuvants herein are designed for optimal use in vertebrates, andin particular humans. Adjuvants herein provide for enhancedimmunogenicity over conventional adjuvants, thereby allowing forvaccines to include smaller quantities of antigen. Adjuvant embodimentsherein are useful with other antigen combinations beyond those useful inthe treatment of growth hormone and/or insulin-like growth factor 1deficiencies. Novel stand-alone adjuvant embodiments are thereforewithin the scope of the present invention but adjuvants herein willpredominately be described in accordance with somatostatin-basedantigen.

In yet another embodiment, vaccines are provided that result inimmunogenicity against somatostatin that results in diminution ofsomatostatin and thereby removal of a proportion of the inhibition thatsomatostatin exerts on growth hormone release and thereby insulin-likegrowth factor 1 release. Vaccine embodiments herein are optimized forboth safety and function, having highly immunogenic somatostatinconstructs in safe and highly effective adjuvant compositions. Vaccinesof the present invention require relatively smaller amounts of antigen(as compared to conventional vaccines), have enhanced storage life, andare lower cost.

Although the present invention is targeted at treatment of growthhormone and/or insulin-like growth factor 1 deficiency in humans,treatment of these deficiencies in other vertebrates is contemplated tobe within the scope of the present invention. For example, dogs and catsshowing signs of obesity, and horses needing repair or replacement ofcartilage can be treated using the compositions and methods describedherein.

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure.

DEFINITIONS

“Amino acid” refers to any of the twenty naturally occurring amino acidsas well as any modified amino acid sequences. Modifications may includenatural processes such as posttranslational processing, or may includebut are not limited to phosphorylation, ubiquitination, acetylation,glycosylation, covalent attachment of flavin, ADP-ribosylation,cross-linking, iodination, methylation, and the like.

“Antibody” refers to a Y-shaped molecule having a pair of antigenbinding sites, a hinge region and a constant region. Fragments ofantibodies, for example an antigen binding fragment (Fab), chimericantibodies, antibodies having a human constant region coupled to amurine antigen binding region, and fragments thereof, as well as otherwell known recombinant antibodies are included in the definition ofantibody in accordance with the present invention.

“Isolation” refers to a polynucleotide or polypeptide that has beenseparated or recovered from at least one contaminant of its naturalenvironment. In some cases the polynucleotide or polypeptides have beenseparated or recovered from 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% of contaminants of its naturalenvironment. Ordinarily, isolated polynucleotides or polypeptides areprepared using at least one purification step. In this regard, purify orpurification, refers to a target polypeptide free from at least 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, 96%, 97% 98% or 99% of thecontaminating polypeptides. Purification of a polypeptide fromcontaminating polypeptides can be accomplished through any number ofwell known techniques, including ammonium sulfate or ethanolprecipitation, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography, and lectinchromatography.

“Obesity” refers to a subject being at least 20% over an ideal bodyweight. For a human, for example, ideal body weight is determined by thesubject's height, age, sex and build. Obesity herein includes the terms:mildly obese (20-40% over ideal weight), moderately obese (40-100% overideal weight) and severely obese (over 100% of ideal weight).

“Patient” refers to a vertebrate, typically a mammal, in need of thecompositions and/or methods of the present invention, for example ahuman in need of weight loss (obese, for example) or horse in needcartilage repair.

“Percent” nucleic acid or amino acid sequence identity describes thepercentage of nucleic acid sequence or amino acid residues that areidentical with a reference polynucleotide or polypeptide. In someinstances, sequences are aligned and gaps introduced to achieve maximumsequence identity. In some instances a computer program is employed tocalculate percent identity, for example, Gap program (Wisconsin SequenceAnalysis package, Version 8 for Unix, Genetics Computer Group,University Research Park, Madison Wis.), that uses an algorithm of Smithand Waterman, 1981, Adv. Appl. Math., 2:482-489 (each of which isincorporated by reference in their entirety herein) or ALIGN-2 sequencecomparison computer program (see WO 00/15796).

“Polynucleotide” refers to a linear sequence of nucleotides. Thenucleotides are either linear sequence of polyribonucleotides orpolydeoxyribonucleotides, or a mixture of both. Examples ofpolynucleotides in the context of the present invention include—singleand double stranded DNA, single and double stranded RNA, and hybridmolecules that have both mixtures of single and double stranded DNA andRNA. Polynucleotides also includes one or more modified nucleotides andpeptide nucleic acids (PNA).

“Protein,” “peptide,” and “polypeptide” are used interchangeably todenote an amino acid polymer or a set of two or more interacting orbound amino acid polymers.

“Treatment” or “treating” refers to improvement of a subject relative toan untreated subject in a relatively identical situation. Treatment ortreating generally indicates that a desired pharmacological and/orphysiological effect has been achieved using the compositions andmethods of the present invention. Treatment or treating can includeprophylactic treatments.

“Vaccine” refers to any composition that can stimulate the vaccinatedsubject's immune system to produce antibodies for the purposes describedherein.

Growth Hormone

Growth hormone is a 191 amino acid peptide produced and released fromsomatotroph cells in the anterior pituitary. Growth hormone levels inthe body are regulated by growth hormone releasing hormone (GHRH) andsomatostatin. GHRH results in synthesis and release of growth hormone(stress, exercise, etc. are also known stimulators of growth hormonerelease) while somatostatin inhibits the release of growth hormone.

Growth hormone (GH) is generally involved in a variety of physiologicfunctions in the body, including: increase of height throughoutchildhood, increase of muscle mass through sarcomere hyperplasia,promoting lipolysis, promoting gluconeogenesis in the liver, andinvolvement in fuel homeostasis. Growth hormone deficiencies typicallymanifest themselves in a number of known disease or physiologic states,including: short stature/growth failure if the deficiency occurs duringchildhood, strength deficits, loss of bone mass, increase incardiovascular risks, e.g., chronic heart failure (Tien et al., GrowthHormone: A Promising Treatment for the Failing Heart, 2000,Pharmacotherapy 20(9):1096-1106, incorporated herein by reference in itsentirety), and other like states. In addition, supplemental growthhormone to a subject is potentially useful in the treatment of wounds,burns, obesity, and the like. Vickers et al., 2002, Adult growth hormonetreatment reduces hypertension and obesity induced by an adverseprenatal environment, J. Endocrinol, 175(3):615-623; Ramirez et al.,1998, Is there a role for growth hormone in the clinical management orburn injuries, Growth Hormone IGF Res. Suppl. B:99-105; and Lal et al.,2000, Growth hormone, burns and tissue healing, Growth Hormone IGF Res.10 Suppl. B:539-543, each of which is incorporated by reference hereinfor all purposes.

Conventional therapies for combating growth hormone deficiency includesupplementation to the afflicted individual with recombinant humangrowth hormone (see, for example, U.S. Pat. Nos. 4,446,235 and4,601,980). Growth hormone supplementation therapies typically requiresubcutaneous injection of recombinant growth hormone on a continualbasis, i.e., daily injections for at least 18 months is typical,although a significant number of individuals require lifetime treatment.Recently, growth hormone supplementation therapies have been used intreatment of Multiple sclerosis (MS), treatment of fibromyalgia,treatment of Crohn's disease and/or ulcerative colitis, treatment toreverse effects of aging, treatment of burns, and treatment foridiopathic short stature. However, treatment with recombinant growthhormone therapy has been shown to potentially increase the risk ofdiabetes, colon cancer, etc. In addition, treatment of a patient with arecombinant protein is typically lacking in internal feed-back controlsproviding an environment of constant monitoring and patient care, withincrease risks associated when the patient has an active malignancy.Further, recombinant growth hormone is extremely expensive to procure,and costs of use over the course of one or more years can beprohibitive, as are the regiment of daily administrations via injection.As such, embodiments of the present invention provide an unexpected andsubstantial benefit in treatment of these growth hormone deficiencyrelated diseases or conditions.

Insulin-Like Growth Factor 1 (IGF-1)

IGF-1 is a polypeptide protein hormone similar in structure to insulin.IGF-1 is produced in the liver and other target tissues in response togrowth hormone and generally has anabolic effects upon release.Typically, IGF-1 acts through the AKT signaling pathway (AKTrepresenting a family of serine/threonine-specific protein kinases).Generally, IGF-1 anabolic effects include cell growth and multiplicationas well as an inhibition of apoptosis at target sites.

A number of factors are involved in influencing IGF-1 levels in apatient's circulation, including: level of growth hormone, geneticmake-up, time of day, age, gender, exercise status, stress, body massindex, and disease state. IGF-1 deficiency can be characterized bygrowth retardation or failure, and is associated with severalconditions, including: obesity, types 1 and 2 diabetes, cardiovasculardisease, various stress disorders, and the like.

Recombinant IGF-1 has been used in the treatment of several of thesemaladies, with mixed results. Presently, Increlex® (a recombinant IGF-1produced by Tercica) is available in the United States for treatment oftarget disorders, although clinical results have varied.

Somatostatin

Somatostatin is a peptide hormone that inhibits, among other things,release of growth hormone from the anterior pituitary. Somatostatinregulates various endocrine functions via interaction withG-protein-coupled somatostatin receptors on target endocrine cells.Somatostatin is secreted from sites in the hypothalamus, stomach,intestine and pancreas. Control of somatostatin levels in a targetanimal has most recently been identified as a point of interest forincreasing productivity of farm animals, i.e., control of somatostatinlevels enhances dairy cow milk production or a farm animals' size, etc(see co-pending application Ser. No. 12/198,579, entitledChloramphenicol Acetyl Transferase (CAT)—Defective Somatostatin FusionProtein And Uses Thereof, which is incorporated herein by reference forall purposes).

In these studies, productivity of farm animals was optimized through useof vaccination protocols with somatostatin antigens. In general, farmanimals immunized with somatostatin had an average daily weight gain of10-20%, an appetite reduced by 9% and an 11% increase in the efficiencyof food utilization. Animals immunized with somatostatin, and also theiroffspring have correct proportions, and the distribution of the weightof the animals between the muscles, bones and fat is the same as incontrol animals (see Reichlin, 1987). However, alternative somatostatintreatments, for example, direct treatment of target animals withanti-somatostatin antibodies, have proven to be overly costly andfunctionally less-dramatic, thereby eliminating direct antibodytreatment as non-practical. Muromtsev G. S., et al., 1990, Basics ofagricultural biotechnology, Agropromizdat, Moscow, pp 102-106. Thesestudies, therefore, indicate that inducing somatostatin immunogenicityin a target animal can accomplish safe and effective results.

The inventor's herein have realized the surprising and unexpected resultthat modification of these vaccination protocols (including methods andcompositions therein) can be used in treatment of human disease orphysiologic states, and in particular, treatment of human growth hormoneand/or IGF-1 deficiency.

Embodiments of the present invention provide somatostatin-basedtreatments for growth hormone deficiency in vertebrates, and, moreparticularly, in mammals. Typical embodiments are directed at treatmentsin humans, dogs, cats and horses. Humans and other mammals immunizedwith somatostatin are treated with vaccines of the invention (see below)to limit or inhibit the effects that native somatostatin has on growthhormone release. For example, where recombinant growth hormone would beadministered to a patient deficient in growth hormone or in need ofexcess growth hormone, for treatment of burns, cardiac therapy,diabetes, etc, vaccines of the invention would be provided to result inadditional endogenous growth hormone release. Vaccine antigens andadjuvants are optimized for vertebrates use and in particular human useand disease treatment. Since somatostatin is highly conserved invertebrates, embodiments of the present invention are useful ineliciting an immune response in all target vertebrates vaccinated usingmethods and compositions herein. A significant benefit of the presentinvention is that vaccinated patients can go weeks to months betweenbooster events, allowing the patient's immune system to limit oreliminate somatostatin from the system.

Embodiments of the present invention also provide somatostatin-basedtreatments for IGF-1 deficiency in vertebrae, and in particular inmammals, e.g., humans, dogs, cats, horses, and the like. Vertebraeimmunized with vaccines of the invention limit or inhibit the effectssomatostatin has on IGF-1 levels in that animal. Treatment embodimentscan be used to treat any number of diseases and/or conditions associatedwith IGF-1 deficiency or where additional IGF-1 is required to improvethe treated animal's health or condition.

As such, aspects of the present invention facilitate somatostatin basedimmunization vaccines by providing highly immunogenic materials for usein prevention and treatment of disease and/or other conditions. Thesesomatostatin based immunization compounds have been optimized forexpression and antigenicity.

In some embodiments, somatostatin-14 is expressed as a codon-optimized,CAT-deficient somatostatin chimeric polypeptide. These materials providean unexpected therapeutic for use in treatment of growth hormone andIGF-1 deficiency based diseases as well as where additional levels ofeither growth hormone or IGF-1 would be useful in a treatment regimen.As described more fully below, the present invention also providesoptimized adjuvants for maximizing the effects of the codon-optimized,CAT-deficient somatostatin chimeric polypeptides. Somatostatin-basedantigens of the invention are designed to provide larger size molecules(27,000+ Daltons vs. 1,600 for native somatostatin), immunogenicity andresistance to degradation. In this manner a somatostatin-based antigenof the invention is present longer (longer ‘A life in the patient), withgreater effect (greater immunogenicity), in a treated patient. Thesenovel antigens for use in vertebrates provide optimal exposure for apatients’ immune system to respond.

Novel Vaccine Embodiments for Use in Treatment of Growth HormoneDeficiency

Somatostatin has two active forms that are produced by alternativecleavage of a polypeptide. Costoff A. Section 5, Chapter 4: Structure,Synthesis, and Secretion of Somatostatin. Endocrinology: The EndocrinePancreas. Medical College of Georgia, page 16, incorporated by referencein its entirety for all purposes. Although it is contemplated thateither somatostatin form can be used in somatostatin-based antigenembodiments herein, somatostatin-14 will be described in detail.Somatostatin-14 is a biologically active tetradecapeptide produced inthe hypothalamus and gastrointestinal tract (stomach, intestine, andpancreas). The amino acid sequence of the tetradecapeptide isAGCKNFFWKTFTSC (SEQ ID NO: 1). The sequence of somatostatin-14 is highlyconserved among vertebrates (Lin X W et al. Evolution of neuroendocrinepeptide systems: gonadotropin-releasing hormone and somatostatin. Comp.Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol. 1998119(3):375-88.) The tetradecapeptide is encoded by a nucleic acidsequence:

(SEQ ID NO: 15) GCTGGCTGCAAGAATTTCTTCTGGAAGACTTTCACATCCTGT(note that other nucleic acid sequences can beused to code SEQ ID NO:1, however, SEQ ID NO: 15is provided for illustrative purposes).

Somatostatin-14 is known to have strong inhibitory effect on a largenumber of hormones involved in the growth and utilization of food inanimals. As previously described in U.S. Pat. No. 6,316,004 and U.S.patent application Ser. No. 12/198,579 (each incorporated herein byreference for all uses), somatostatin and chimeric versions ofsomatostatin can be used in immunization of animals for increase indaily weight and, where appropriate, milk production. These immunizationprocedures were performed with conventional adjuvants. Somatostatin-14immunization has not been used in the treatment of any particular growthhormone deficiency state.

One aspect of the invention provides isolated nucleic acid moleculesthat encode chimeric proteins having optimized somatostatin immunogenicactivity. In particular, embodiments of the invention include novelnucleic acid constructs that encode CAT fusion proteins havingimmunogenic activity for somatostatin. These polypeptides have beenidentified for optimal functional activity in immunization proceduresand use in treatment of growth hormone and/or insulin-like growth factor1 deficiencies and in particular in treatment of mammalian growthhormone and/or insulin-like growth factor 1 deficiencies.

In one embodiment, a construct having a schematic as shown in FIG. 1 isprovided to encode the chimeric polypeptides of the invention. Nucleicacid constructs of the invention generally encode an inactive CAT enzymewithout 10 C-terminal amino acids and includes one or two histidinesreplaced amino acids. The CAT enzyme is inactivated by removing theimidazole group of His 193 (His 195 in the canonical CAT_(III) variant).In another embodiment the CAT enzyme is inactivated by removing theimidazole groups of both His 193 and the nearby His192 (respectivelyHis195 and His194 for CAT_(III) Removal of the essential His 193 (His195in CAT_(III)) imidazole group from the active site of CAT andreplacement with an alanine, glycine or other like amino acid results insubstantial inactivation of the CAT enzyme (see for example, Lewendon Aet al. (1994) Replacement of catalytic histidine-195 of chloramphenicolacetyl transferase: evidence for a general base role for glutamate.Biochemistry. 33(7):1944-50; White et al., (2000) Characterization ofChloramphenicol and Florfenicol Resistance in Escherichia coliassociated with Bovine Diarrhea. J. Clin. Micro 38(12) p 4593-4598. eachof which is incorporated by reference herein for all purposes). Finally,embodiments herein can also include CAT enzyme inactivation throughremoval of the imidiazole group of His 192 alone (His 194 for CAT₁₁₁).As for His193, replacement can be with an alanine, glycine or other likeamino acid.

In some aspects, the one or more replaced histidine amino acids areencoded by nucleic acids located at position numbers 574-576 and 577-579of SEQ ID NO: 2 (corresponding to amino acid numbers 192 and 193 in SEQID NO: 3). In some embodiments the nucleic acid sequences of theinvention include SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Chimericproteins of the invention that include the histidine replaced constructsherein provide highly immunogenic proteins with little or no CATactivity, a significant improvement over the existing art. Theinactivated CAT enzyme embodiments are attached to a somatostatinpolypeptide of the invention. This attachment can be made directly orwith a linker (as described more fully below).

CAT inactivation, at sites his192 and his193, can be accomplished viaany number of known procedures to those skilled in the art includingsite-directed mutagenesis and synthetic gene assembly. In oneembodiment, the nucleic acid sequence that encodes histidine 193 orhistidine 192 are modified to encode an alanine, glycine or other likeamino acid. In another embodiment, the nucleic acid sequences thatencode both histidine 192 and 193 are modified to encode alanine,glycine or other like amino acids. Typical replacements for the 192 and193 chimeric polypeptide include: alanine, alanine, alanine, glycine,glycine, alanine, glycine, glycine.

Embodiments of the present invention also include the amino acidsequences for CAT deficient polypeptides of the invention, includingamino acid sequence having SEQ ID NO: 7, 8 and 3 (corresponding tohis->gly at 193, his->ala at 193, and his->gly at both 192 and 193).

The realization that CAT enzyme could be inactivated and used as acarrier protein for presentation of somatostatin-14 in the treatment ofdiseases and/or conditions herein, especially in mammals, was anunexpected finding of the inventors. Non-inactivated CAT has beendescribed as the enzyme responsible for plasmid mediated bacterialresistance for both Chloramphenicol and Florfenicol (fluorinatedanalogue) in multiple, worldwide, gram-negative bacterial isolates. Theuse of the non-inactivated CAT, as described U.S. Pat. No. 6,316,004B1,predates these scientific discoveries. As such, according to currentunderstanding and established standards, the use of non-inactivated CATwould be counterindicated due to safety concerns (i.e., creation of moreantibiotic resistant hosts). Chloramphenicol was discovered about 60years ago and was primarily used as an antibiotic. Several healthconcerns arose out of this usage including recipients of the drugdeveloping aplastic anemia. In addition, where the antibioticfluorinated analogue continued to be used, for example in the cattleindustry, an increase in several strains of bacteria becoming resistantto the antibiotic, due to plasmid encoded genes. AlthoughChloramphenicol continues to be used in eye drops to treat bacterialconjunctivitis, it is not used in the United States to treat otherbacterial borne disease. As such, the realization and development ofusing an inactivated CAT enzyme in any matter in mammalian, and inparticular human, is surprising, where utilization of the carrierprotein based benefits described throughout this specification, whileavoiding the significant health concerns of active CAT, providessignificant improvement to vaccines described herein.

Note that these “carrier” related improvements of CAT for use with smallmolecules are discussed in co-pending and related U.S. PatentApplication S/N PCT/US08/68195 as well as in U.S. Pat. No. 6,316,004both of which are incorporated by reference herein for all purposes. Inparticular, the inventors herein unexpectedly found that an inactivatedCAT enzyme as a carrier protein for somatostatin-14 could avoid thesignificant health risks associated with the enzyme while utilizing thechimeric proteins enhanced capacity for immunogenicity, resistance toenzyme degradation, increased half-life and enhanced uptake by thepatient's macrophages.

As shown in FIG. 1, the non-active CAT enzyme can be linked tosomatostatin-14 via a variable length linker or spacer. The spacer isrequired to insure presentation of the encoded somatostatin on a globalsurface. Spacer embodiments herein provide for optimal proteaseresistance and for optimal epitope exposure and have shown unexpectedimprovement over constructs not having the linker sequence(s) of thepresent invention.

Spacer embodiments, therefore, have been optimized in length andcomposition to ensure CAT-defective somatostatin expression in variousmicroorganisms, and in particular in E. Coli. Original constructs asdescribed in U.S. Pat. No. 6,316,004, included a spacer having rare E.Coli codons and required the co-expression of rare tRNAs from a secondor helper plasmid. Spacer embodiments herein remove these rare E. Colicodons and thereby remove the need for a second helper plasmid, animprovement over previous technology.

In typical embodiments, the spacer has a nucleic acid sequence oftgggaactgcaccgttctggtccacgcccgcgccctcgcccacgtccggaattcatg (SEQ ID NO:9). One example of a spacer of the invention has an amino acid sequenceof welhrsgprprprprpefm (SEQ ID NO: 10). A typical amino acid sequencefor a spacer of the invention is welhrsgp(rp)_(n)efm where n>1 (SEQ IDNO: 11). As noted above, these novel spacer sequences provide forenhanced protease resistance (thereby allowing for increased productionas compared to constructs disclosed in U.S. Pat. No. 6,316,004) andoptimal somatostatin-14 exposure. This combination of somatostatinattaches to an inactivated CAT enzyme by an optimally configured linkershow unexpected improvement when used to immunize target patients fordisease treatment. These constructs are used as antigens in thetreatment of growth hormone and insulin-like growth factor 1 deficiencybased diseases.

Further, these chimeric constructs show enhanced storage stability ascompared to somatostatin-14 alone. In addition, the somatostatin-basedantigens of the present invention provide for greater half-life in thepatient given the enhanced resistance to degradation in these materials.It is noted that other carrier polypeptides can replace inactivated CATas attached to the somatostatin. For example, somatostatin-14 can becombined with KLH, tetanus toxoids or CRM instead of inactivated CATenzyme.

Embodiments of the invention also provide novel adjuvant compositionsfor enhanced induction of humoral immunity in a target patient. Theseadjuvant compositions provide a significant improvement overconventional materials for the induction of a humoral response and aresafe for use in human targets. Adjuvant compositions herein are usedwith somatostatin-based antigens to produce vaccines of the invention.Vaccines of the invention are then useful in the treatment of GH and/orIGF-1 deficiency-based diseases or conditions.

In embodiments herein, all components of adjuvant compositions are ofnon-animal origin, thereby eliminating potential cross-contamination ofvaccinated humans from potentially contaminated adjuvant components. Forexample, embodiments herein can utilize animal origin free Tween 80.Surprisingly, animal origin free Tween 80 shows significantly betterresults in the use of vaccines herein as compared to animal origin Tween80, and eliminates the possibility of animal-based contamination intothe vaccine, e.g., Bovine Spongiform Encephalopathy (BSE). In addition,animal origin free Tween 80 shows better capacity to emulsify ascompared to animal origin Tween 80, providing an additional unexpectedbenefit for its use in accordance to embodiments herein.

Adjuvant embodiments herein are also free of benzene and other likecarcinogenic compounds. These embodiments provide a safety benefit notavailable in most conventional adjuvant compounds. For example,embodiments herein utilize Carbopol 974P or benzene free polycyclicacid.

In one embodiment, the immunologic adjuvant comprises a carbopol base, asqualene base and an arabinogalactan solution. In more detail, theCarbopol base is prepared using Carbopol 974P in water or saline. Thesqualene base is prepared from a combination of squalene, non-animalorigin Tween 80 and Span 85. In some embodiments the squalene base isMF59 (Chiron Corp., Emeryville, Calif.). The arabinogalactan isdissolved in PBS or saline. Adjuvant compositions are combined withchimeric polypeptides of the invention to produce vaccines of theinvention.

In yet another embodiment, the immunologic adjuvant comprises a Carbopolbase, a squalene base and a tragacanthin solution. In more detail, theCarbopol base is prepared using Carbopol 974 P in water or saline. Thesqualene base is prepared from a combination of squalene, non-animalorigin Tween 80 and Span 85. Purified tragacanthin is dissolved in PBSor saline. Adjuvant compositions are combined with chimeric polypeptidesof the invention to produce vaccines or the invention.

Specific adjuvant combination and concentrations are shown in Example 3.Adjuvants in accordance with the present invention are safe andeffective for human use, avoid animal products, avoid petroleum basedhydrocarbons, and avoid carcinogenic compounds.

Vectors and Host Cells

The present invention also relates to vectors comprising thepolynucleotide molecules of the invention, as well as host cellstransformed with such vectors. Any of the polynucleotide molecules ofthe invention may be joined to a vector, which generally include aselectable marker and origin of replication, for the propagation host ofinterest. Host cells are genetically engineered to include these vectorsand thereby express the polypeptides of the invention. Generally,vectors herein include polynucleotides molecules of the inventionoperably linked to suitable transcriptional or translational regulatorysequences, such as those for microbial or viral host cells. Examples ofregulatory sequences include transcriptional promoters, operators, orenhancers, mRNA ribosomal binding sites, and appropriate sequences whichcontrol transcription and translation. Nucleotide sequences are operablylinked when the regulatory sequences herein functionally relate to thechimeric polypeptide encoding polynucleotides of the invention.

Typical vehicles include plasmids, yeast shuttle vectors, baculovirus,inactivated adenovirus, and the like. In one embodiment the vehicle is amodified pET30b CatSom plasmid (see FIG. 1). Target host cells for useherein include bacterial host, e.g., E. Coli., yeast, SF-9 insect cells,mammalian cells, green plants, and the like.

In one embodiment, the regulatory sequences include a T7lac, CAT, Tip,or T5 promoter for expression of the chimeric polypeptides of theinvention in E. coli or other like microbes. These regulatory sequencesare known in the art and are used under appropriate and knownconditions.

Various plasmids of the invention have been constructed for expressionof chimeric polypeptides of the invention through utilization of targetregulatory sequences. Illustrative plasmids can include a T7lac promoter(see FIG. 1).

Host cells for expression of target chimeric polypeptides includeprokaryotes, yeast and higher eukaryotic cells. Illustrative prokaryotichosts include bacteria of the genera Escherichia, Bacillus, andSalmonella as well as the genera Pseudomonas and Streptomyces. Intypical embodiments the host cell is of the genera Escherichia and canbe Escherichia Coli (E. coli). Host cells (yeast or bacterial) can alsobe used to produce target polypeptides of the invention as virus-likeparticles (VLPs).

As shown in the Examples below, constructs of the invention provide foroptimal CAT deficient somatostatin expression under a variety ofconditions. These constructs are particularly efficient for expressionin prokaryotic hosts and in particular bacteria of the generaEscherichia.

Endotoxin Free Fusion Protein for Use in Vaccines of the Invention

Aspects of the present invention include use of endotoxin free,codon-optimized, CAT-deficient somatostatin. In one embodiment, thechimeric immunogenic somatostatin-comprising proteins of the inventionare prepared by transforming target cells with appropriatesomatostatin-containing vehicles. As noted above, vehicles for useherein include known plasmid and vector systems suitable for expressionin selected target cells.

In an aspect of the invention, chimeric immunogenicsomatostatin-comprising proteins are expressed in target host cells.Chimeric protein expression is performed using target regulatorysequences. In some aspects the chimeric polypeptides have been optimized(especially with regard to spacer sequences disclosed herein) forexpression in E. Coli.

Chimeric protein can then be purified in accordance with known proteinpurification technologies, including, for example, lysozyme lysis,differential centrifugation of inclusion bodies, sieve chromatographyand the like. Refolding procedures can be conducted in guanidinechloride and urea at alkaline pH followed by dialysis andlyophilization.

In one embodiment, E. coli cells are transformed using thecodon-optimized, CAT-deficient somatostatin containing plasmid—pET30bCatSom; the pET30b CatSom having appropriate E. Coli base regulatorysequences for expression. In some cases, fermentation of approximatelyten liters of these cells provides at least 500 grams and in some cases600 grams of total biomass, yielding about 4-6 grams of total protein.It is estimated from silver and Coomassie blue staining that about halfof the protein is the target chimeric protein (see Example 2 and FIG.2).

In some embodiments herein, chimeric protein of the invention ispurified from transformed host cells in a substantially endotoxin freestate. Additional purification will remove or lower the endotoxin toacceptable levels for human uses according to Food and DrugAdministration standards.

As such, some embodiments herein are directed at production ofsubstantially endotoxin free chimeric proteins for use in vaccines. Incertain embodiments the endotoxin levels are at or below 1 EU/ml and inother embodiments the endotoxin levels are substantially eliminated,i.e., the chimeric polypeptides of the invention are substantiallyendotoxin free.

In one embodiment, recovered IB from lysed host cells was washedmultiple times using a wash buffer devoid of endotoxin. The recovered IPpellet can optionally be washed until endotoxin levels are belowapproximately 1 EU/ml (endotoxin tests can be performed using one ormore known assays, including commercially available test kits from MPBiochemicals, Charles River, etc.). In some embodiments the wash bufferis endotoxin free and includes one or more proteolytic proteininhibitor(s), e.g., phenylmethanesulphonylfluoride (PMSF). In someembodiments the wash buffer is phosphate buffered saline (PBS) having aninhibitory effective amount of PMSF and/or Aminoethyl-BenzenesulfonylFlouride Hydrochloride (AEBSF).

In some aspects, substantially endotoxin free pellets can be treatedwith a protein unfolding solution at pH 12.5 containing urea andrefolded in a protein refolding solution containing a reduced molarityof urea with arginine, glycerol and/or sucrose. Purified chimericprotein concentration is modified to be between 1 and 3 mg/ml andtypically about 1.4 to 1.8 mg/ml. In some cases, substantially endotoxinfree chimeric protein is provided to vaccine formulations at about 1.5to 5 mg/2 ml dose and more typically from 2.0 to 3.5 mg/2 ml dose. Otherendotoxin removal procedures can also be utilized, for examplecommercially available ion-exchange endotoxin removal columns,hydrophobic columns, etc.

Vaccines

Vaccines of the invention are combinations of immunologic adjuvants asdescribed herein and target antigens useful in the prevention ortreatment of a human disease state.

The pharmaceutical dosage for vaccine embodiment herein includes 1-5 mgchimeric polypeptide. In all embodiments herein the vaccine should besterile, fluid and stable under conditions of manufacture and storage.The prevention of the action of microorganisms can be accomplished byaddition of various antibacterial and antifungal agents, for example,parabens, chlorobutanol, sorbic acid, thimerosal and the like.

Vaccines herein typically include an antigen in a total protein amountof from about 1 mg/2 ml to 3 mg/2 ml dose, wherein approximately 5% to25% of the dose is adjuvant and more typically from about 10% to 20% ofthe dose is adjuvant. In some embodiments the adjuvant makes-up about18% of the dose.

For purposes of illustration, the adjuvants of the invention arecombined with a somatostatin based polypeptide to provide a vaccineuseful in the treatment of human growth hormone and/or insulin-likegrowth factor 1 deficiency based diseases and/or conditions. Note thatthe adjuvants described herein could be combined with other antigens toproduce novel vaccines useful in the treatment of other target humandisease.

Method for Treatment of Human Disease

The invention provides pharmaceutical grade vaccines containing chimericpolypeptides and adjuvants of the invention. Such vaccines can beadministered to patients having a growth hormone and/or insulin-likegrowth factor 1 deficiency to facilitate appropriate release fromendogenous sources in the patient.

Vaccines of the invention are provided to patients having a growthhormone and/or insulin-like growth factor 1 deficiency. In oneembodiment, vaccines of the invention provided 1 to 2 times, with 3 to 5boosters over the course of a treatment. A typical vaccine antigenamount is from 1 to 5 mg/ml chimeric polypeptide. Vaccines can beadministered by known techniques. In one embodiment the vaccine isadministered via subcutaneous injection. In another embodiment thevaccine is administered by intradermal injection, intramuscularinjection or infusion.

Vaccine embodiments of the invention can further include dispersing orwetting agents, suspension agents, or other like materials. For example,embodiments can include sterile oils, synthetic mono- or diglycerides,fatty acids or oleic acids.

Vaccines are typically prepared as sterile, aqueous solutions. Thesesolutions are stable under conditions of manufacture and storage. Insome aspects, additional agents can be included in the vaccine toprevent microorganism action, for example, antibacterial or antifungalagents.

Vaccine solutions of the invention are prepared by incorporating thematerials in the required amounts (antigen, adjuvant, other ingredients)and can be followed by terminal sterilization, e.g., via UV light orozone treatment. Alternatively, vaccine solutions of the invention canbe prepared using individually sterilized components prior to finalassembly (in which case no terminal sterilization is required).

Treatment progress for patients receiving vaccine embodiments of theinvention can be monitored and additional administrations provided.Increase in growth hormone levels, increase in insulin-like growthfactor 1 levels, and functional benefits (for example, acceptable weightloss in an obese patient) are all targets for monitoring of treatmenteffectiveness. In addition, where type 1 or 2 diabetes or obesity isbeing treated, blood glucose, IGF-1 levels, lipid profiles, insulinlevels, and Hemoglobin Bound A1c (HbA1c) levels can be monitored todetermine effectiveness of treatment on a patient. Based on anindividual patients' progress, additional vaccine injections can beperformed using more or less antigen in accordance with the presentinvention. In addition, alternative adjuvant combinations may be used tomodify a particular patients' response to vaccination, as determined bythe health care professional.

Embodiments herein can be combined with other conventional therapies forthe target growth hormone and/or insulin-like growth factor 1 deficiencydisease state or condition. For example, vaccinations of the inventioncan be combined with replacement insulin in the treatment of type 1diabetes, or vaccinations can be combined with weight loss surgery orlow calorie diets in a patient suffering from severe obesity.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Construction of CAT-defective Somatostatin Fusion Protein

The present example illustrates the production of a CAT-defectivesomatostatin fusion protein in accordance with embodiments of thepresent invention. Site-directed mutagenesis was performed on plasmidpET30b-Cat-Som to replace His192 and His193 with glycine residues (aftermodification: Gly192 and Gly193). Inactivation of the His193 (and His192) residues eliminates the capacity of the CAT enzyme to acceptprotons, thereby providing complete inactivation of the CAT.

The spacer in the same pET30b-Cat-Som (having the His replacement(s))was codon-optimized for expression by E. coli in the absence ofco-expressed tRNA molecules.

The modified CAT-defective somatostatin nucleic acid construct is shownas SEQ ID NO: 12. The CAT-defective somatostatin fusion protein sequenceis disclosed as SEQ ID NO: 13, being compared to an unmodifiedCAT-somatostatin fusion protein (SEQ ID NO: 14).

Example 2 CAT-Defective Somatostatin Fusion Protein can be Expressed atHigh Levels

The codon-optimized CAT-defective somatostatin construct as described inExample 1 was used to express the fusion protein in BL21(DE3) cells.Transformed cells were grown in LB and induced with 0.4 mM IPTG forapproximately three hours. One milliliter of cells from a density of OD0.7 culture were pelleted, and heated at 70° C. for ten minutes in 100μl SDS sample buffer. A sample of 40 μl of cell extract was loaded perlane for SDS PAGE.

As shown in FIG. 2, a 28 KD band corresponding to the predicted size ofa codon-optimized, CAT-defective somatostatin fusion protein was visiblein lanes 1 (LB+IPTG, reduced) and 3 (LB+IPTG) after induction with IPTG.No expression is seen in control lanes 2 (LB, reduced) and 4 (LB). Asexpected, there was no difference in fusion protein size when run understandard or reducing conditions.

Example 3 Endotoxin Free, Codon-Optimized CAT-Deficient SomatostatinContaining Vaccine

An illustrative vaccine in accordance with the present invention:

Reagent Solution:

1. Carbopol Base

-   -   a. Dissolve 0.5 grams of Carbopol 974P in water or saline    -   b. Mix and boil to dissolve. Followed by autoclaving.    -   c. Store at 4° C.

2. Squalene Base

-   -   a. Mix 58.1 ml of squalene, 4.6 ml of non-animal origin Tween 80        and 5.2 ml of Span 85.    -   b. Mixture was filtered through a 0.2μ filter.    -   c. Store at 4° C.

3. Tragacanthin Solution

-   -   a. Extract tragacanth gum with methanol.    -   b. Collect methanol insoluble fraction.    -   c. Dry at room temperature.    -   d. Store at room temperature in a desiccated state,    -   e. Add 1 gram of dried Tragacanthin in water or saline.    -   f. Mix and boil to dissolve, followed by autoclaving.    -   g. Store at 4° C.

Vaccine Preparation

-   -   1. Vaccine antigens are prepared in saline or PBS at 5 mg/ml or        lower.    -   2. Add 6.79 ml of squalene base to mixing bottle.    -   3. Add 10 ml of Carbopol base to Squalene base. (CS)    -   4. Mix well.    -   5. Add 10 ml of Tragacanthin solution to CS solution.    -   6. Mix well.    -   7. Vaccine antigens, undiluted or diluted to use in saline or        PBS, are added to a final volume of 82 ml.    -   8. 1 ml of a 1% Thimerosal solution is added and mixed well.    -   9. Store vaccine at 4° C. until use.        Alternative illustrative vaccine in accordance with the present        invention:

Reagent Solutions:

1. Carbopol Base:

-   -   a. Dissolve 0.5 grams of Carbopol 974P in water or saline;    -   a. Mix and boil to dissolve; and autoclave    -   b. Store at 4° C.

2. Squalene Base:

-   -   a. Mix 58.1 ml of squalene, 4.6 ml of non-animal origin Tween 80        and 5.2 ml of Span 85; and filter through 0.2μ filter    -   b. Store at 4° C.

3. Arabinogalactan Solution:

-   -   a. Add 1-10 grams of arabinogalactan into PBS or saline;    -   b. Mix and boil to dissolve; and autoclave    -   c. Store at 4° C.

Vaccine Preparation:

-   -   1. Vaccine antigens are prepared in saline or PBS at 5 mg/ml or        lower;    -   2. Add 6.79 ml of squalene base to mixing bottle;    -   3. Add 10 ml of Carbopol base to the Squalene base;    -   4. Mix thoroughly and add 10 ml of arabinogalactan solution;    -   5. Antigens of the invention, undiluted or diluted, to use in        saline or PBS, are added to a final volume of 82 ml.    -   6. 1 ml of a 1% thimerosal solution is added and the vaccine        mixed; and    -   7. The vaccine stored at 4° C. until use.

Example 4 Treatment of Cardio Vascular Disease Using Vaccine of Example3

The present Example uses rats with left ventricle dysfunction asprepared in the protocol published in Genentech, 1995. Two groups ofrats are segregated (each member of each group having a ligated leftcoronary artery), a first treatment group receives the vaccinations ofthe invention and a second control group (no vaccination, but otherwisetreated the same). Each member of the treatment group receives avaccination and then 21 days later a second vaccination, administeredintramuscularly (1 ml/dose). Serum IGF-1 levels and anti-somatostatinantibodies are measured at day 0, day 21 and day 42. At day 42,hemodynamic parameters are also measured in both groups as well as adetermination of infarct size and cardiac index.

It is anticipated that the rat group receiving the two vaccinations, asdescribed in Example 3, would have substantially improved cardiacfunction (decrease infarct size and improved cardiac index) as comparedto the control group.

Example 5 Treatment of Obesity Using Vaccine of Example 3

A rat obesity model as described in Vickers et al (2001) will be used todetermine the effect that vaccines of the invention have on obesity.Rats are fed a hypercaloric diet for 30 to 60 days. Hypercaloric ratswill be weighed and separated into three groups—a saline control groupand a vaccination group continued on a hypercaloric diet, and a normalcaloric diet group. Rats receiving the vaccination will receive 2×1 mldose intramuscularly at day zero and at day 21. All rats will be weighedweekly throughout the study period. At day 42, all rats will be weighedand serum collected for IGF-1 analysis, urea analysis andanti-somatostatin antibody levels.

It is expected that rats receiving the vaccinations as described inExample 3 will have substantially improved weight control over salinecontrol groups and show corresponding serum results that correlate thatweight control is due to growth hormone modification. The vaccinatedgroup will show substantially the same weight control as the normalcaloric intake group.

Example 6 Treatment of Growth Deficiency Using Vaccine of Example 3

Three week old Cox (CD) rats will be vaccinated monthly for 3 monthsusing a 1 ml dose. Each vaccination will occur intramuscularly orsubcutaneously. Control rats will receive saline injections, using thesame mode of administration and the same volume of material foradministration. All rats will be weighed to determine growth on a weeklybasis and bled at 0, 4, 8, 12 and 16 weeks. Serum will be collected at asimilar schedule and analyzed for IGF-1, urea and anti-somatostatinantibody levels.

It is expected that CD rats receiving vaccinations as described inExample 3 will have substantially improved growth as compared to controlCD rats. Treated rats should show serum results that confirmvaccinations effects on treated rats.

Example 7 Increased Somatostatin Leads to Weight Gain in Mice

Mouse obesity studies were performed to see if exogenously administeredsomatostatin would cause weight gain in mice. A seven day study wasperformed on outbred mice where three different inventive adjuvants werecombined with recombinant somatostatin (adjuvants are referred to asJH14, JH17 and JH18). Each adjuvant was spiked with 1 mg/mlrecombinately produced somatostatin (produced in 2009) and one adjuvant(JH14) was also spiked with 2007 produced somatostatin at aconcentration of 2.57 mg/ml. A control group of mice received a sterilesaline injection.

Each mouse was weighed at the start of the study to determine a baselineweight. Vaccinations on each mouse were performed on day 0 by IPinjection of 0.5 ml of vaccine. Somatostatin in JH17 and 18 adjuvantswere administered to female mice and JH14 to male mice. Mice were fed anormal diet over the course of the 7 days with each mouse beingre-weighed at day 7.

As shown in FIG. 3, outbred mice receiving somatostatin remained healthyand showed little adverse effects from the one week study. Female micein the JH17 group had an increase in weight, while the female JH18 micewere similar in weight gain to control mice. Male mice in the two JH14groups (2007 and 2009) both showed weight gain over control group malemice. Overall the findings in FIG. 3 illustrate that the JH14, JH17 andJH18 adjuvants are safe for use in mice, highly consistent and capableof storage, and that exogenously administered somatostatin generallyresults in weight increase in a target mouse, providing evidence thatsomatostatin is involved in weight gain. All of the effects occurredwithin several days of IP immunization. This is due to direct macrophageprocessing and antigen and presentation B lymphocytes at an acceleratedrate, due to route of administration, the nature of the chimericpolypeptides and the adjuvant effect.

The JH14, 17 and 18 Preblends are as described in this and USApplication S/N PCT/US08/68195, entitled Chloramphenicol AcetylTransferase (CAT)-Defective Somatostatin Fusion Protein and UsesThereof. The final formulation had 1 mg protein antigen per 1 ml dose.For this work the following were the formulations:

JH17

Refolded protein 12 ml (5.86 mg/ml)

Dulbecco's PBS 24.5 ml JH 17 Preblend 13.4 ml 1% Thimerosal 0.5 ml

37% formaldehyde 0.1 ml

JH18

Refolded protein 12 ml (5.86 mg/ml) Dulbecco's PBS 24.5 ml JH 18Preblend 13.4 ml 1% Thimerosal 0.5 ml 37% formaldehyde 0.1 ml

JH14

Refolded protein 12 ml (5.86 mg/ml) Dulbecco's PBS 27.8 ml JH 14Preblend 10 ml 1% Thimerosal 0.5 ml 37% formaldehyde 0.1 mlNote that formulations above include thimerosal and formaldehyde tolimit contamination of the vaccines. These materials are only used wheremulti-dose re-entry bottles for dispensing vaccine are in use, typicallyhuman and animal treatment protocols will be packaged in single-usevials, thereby eliminating the need for these preservatives. Also notethat a higher level of antigen was used in the 2007 JH14 lot to make upfor protein degradation.

Example 8 Treatment of Obesity in Mice

Mouse obesity studies were performed using mice from JacksonLaboratories, Bar Harbor, Me. A number of inbred mice from line C57BL/6Jwere obtained from Jackson Laboratories, the mice were: male, showedinduced severe obesity, had polygenic genetics, and exhibited matureonset obesity. In previous testing, Jackson Laboratories had determinedthat this particular strain of mice, when fed on a high fat diet,develops metabolic syndrome phenotypes very similar in nature to thosereported in the human population. For example, C57BL/6J mice fed a highfat diet will show visceral adiposity, insulin resistance,hyperinsulinemia, hyperleptinemia, leptin resistance and hypertension.

Studies were conducted to test the effectiveness of vaccine embodimentsherein for treating obesity, i.e., including limiting weight gain insome mice to causing weight loss in C57BL/6J mice. Six week old micewere fed a 60% kcal % fat diet for 6 weeks. Twelve week old mice werethen broken into one of four groups: group 1 included mice treated withJH14 containing vaccine; group 2 included mice treated with JH17containing vaccine, group 3 included mice treated with JH18 containingvaccine, and group 4 included control mice that were treated with PBSrather than any type of anti-somatostatin type antigen. Mice in eachgroup were vaccinated using a 0.5 ml of the specified vaccine or PBS viaan IP route. After twenty two days the mice were treated again with asecond IP dose using 0.1 ml of vaccine.

Throughout the course of the study (6 weeks) each mouse was weighed twotimes per week and food intake monitored, i.e., to ensure that weightchanges were not due to loss or increase in food intake (see FIG. 4showing cumulative food intake within each of the 4 groups). A terminalbleed was performed on each mouse at the conclusion of the study andIGF-1 levels determined (IGF-1 plasma levels were determined usingDiagnostic Systems Laboratories Inc. Active Mouse/Rat IGF-1 ELISA(DSL-10-29200).

As shown in FIGS. 5 and 6 and Table 1, mice treated with JH14, 17 and 18all showed a highly significant difference (p<0.0001) by parametric ornon-parametric statistical analyses) in percent Final Body Weight vs.Baseline Weight. Significant weight loss was observed in each vaccinatedgroup within the first 7 days while the control group showed sleightweight gain over the same time period. A small weight loss was alsoobserved after the second dose of vaccine (⅕^(th) dose provided onday 1) was administered to the JH14, JH17 and JH18 groups at day 22.

Data from the mouse obesity study provided the following conclusions:(1) although there is not a statistically significant difference betweenJH18 and the controls, in terms of IGF-1 ng/ml, there is a highlysignificant difference between these groups (P<0.0001) by parametric ornon-parametric statistical analysis in percent Final Body Weight versusBaseline Weight; (2) In percent Final Body Weight versus BaselineWeight, JH17 versus the controls produced a statistically significantdifference by both statistical tests; (3) JH18 (which had a mean IGF-1level of 135.8 ng/ml more than JH17), demonstrated a statisticallysignificant difference versus JH17 in percent baseline weight (only bythe non-parametric test); (4) chimeric-somatostatin antigen of theinvention in both JH17 and JH18 adjuvants induced a statisticallysignificant difference in percent Final Body Weight versus BaselineWeight; (5) JH18 was statistically significant when compared with JH17by non-parametric analysis in terms of percent Final Body Weigh versusBaseline Weight; (6) IGF-1 levels can be correlated with a greaterweight loss at the end of the study versus both controls and JH17vaccinates (see Table 2); (7) since all vaccinates had the same doseamounts of the chimeric-somatostatin antigen of the invention, anadjuvant affect was observed within the study; (8) inbred C57BL/6J malemice fed 60% kcal fat diet demonstrated a significant weight loss withinthe first week post IP vaccination; and (9) the weight loss shown hereinpersisted even while the mice ate a 60 kcal % fat diet for the durationof the study.

TABLE 1 Final Body Weight versus Baseline Weight Mann Unpaired %Standard Whitney t-test Group # Baseline Deviation (two tailed)(two-tailed) Controls 10 115.5 6.3 Not Done Not Done JH17 10 107.1 4.7 P= 0.0021 P = 0033   JH18 10 104 3.0 P < 0.0001 P < 0.0001 JH17 vs. JH18— — — P = 0.0355 P = 0.1016

TABLE 2 IGF-1 Statistical Analysis Mean IGF-1 Standard Mann WhitneyGroup # (ng/ml) Deviation (1 tailed) Controls 10 365.6 88.7 Not DoneJH17 10 304.2 99.2 P = 0.0827 JH18 10 440.4 103.7 P = 0.105 

It is understood for purposes of this disclosure, that various changesand modifications may be made to the invention that are will within thescope of the invention. Numerous other changes may be made which willreadily suggest themselves to those skilled in the art and which areencompassed in the spirit of the invention disclosed herein and asdefined in the appended claims. The specification contains numerouscitations to patents and publications. Each is hereby incorporated byreference for all purposes.

What is claimed is:
 1. A method for treating a growth hormone deficiencyin a patient, the method comprising: administering an immunogenic amountof a vaccine to the patient, the vaccine comprising (a) a chimericpolypeptide of somatostatin-14 attached to an inactivatedchloramphenicol acetyl transferase (CAT) enzyme and (b) an adjuvant,wherein the administered vaccine results in an increase in growthhormone levels in the patient having the growth hormone deficiency. 2.The method of claim 1 wherein the vaccine comprises a chimericpolypeptide of somatostatin-14 functionally attached to a substantiallyinactivated CAT enzyme.
 3. The method of claim 2 wherein the adjuvantcomprises an effective amount of Carbopol 974P.
 4. The method of claim 3wherein the adjuvant further comprises squalene and non-animal originTween
 80. 5. The method of claim 1 wherein the patient is an adulthaving growth deficiency.
 6. The method of claim 1 wherein the patientis a child having growth deficiency.
 7. The method of claim 1 whereinthe patient being treated for a growth hormone deficiency has a mucosaldisease.
 8. The method of claim 1 wherein the patient being treated fora growth hormone deficiency has a cardiac disease.
 9. The method ofclaim 1 wherein the patient being treated for a growth hormonedeficiency is obese.
 10. The method of claim 1 wherein the patient is anobese dog.
 11. The method of claim 1 wherein the patient is an obesecat.
 12. The method of claim 1 wherein the patient is a horse in need ofcartilage repair.
 13. A method for treating an insulin-like growthfactor 1 deficiency in a patient, the method comprising: administeringan immunogenic amount of a vaccine to the patient, the vaccinecomprising a somatostatin-based antigen and an adjuvant, wherein theadministered vaccine results in an increase in insulin-like growthfactor 1 levels in the patient having the insulin-like growth factor 1deficiency.
 14. The method of claim 13 wherein the patient being treatedfor insulin-like growth factor 1 deficiency has type 1 or type 2diabetes.
 15. The method of claim 13 wherein the patient being treatedfor insulin-like growth factor 1 deficiency has a stress disorder. 16.The method of claim 13 wherein the patient being treated forinsulin-like growth factor 1 deficiency has cardiac disease.
 17. Anadjuvant for use in a vaccine for treating a vertebrate, the adjuvantcomprising: a Carbopol 974P base; a squalene base; and anarabinogalactan solution; wherein the adjuvant acts as a carrier for asomatostatin-based antigen for treatment of human growth hormone orinsulin-like growth factor 1 deficiency in the vertebrate.
 18. Theadjuvant of claim 17 wherein the squalene base is a combination ofsqualene, non-animal origin Tween 80 and Span
 85. 19. The adjuvant ofclaim 17 wherein the somatostatin-based antigen is somatostatin-14linked to a substantially inactivated CAT enzyme.
 20. The adjuvant ofclaim 17 wherein the inactivated CAT enzyme has one or more wild typehistidine residues replaced with alanine, glycine or other like aminoacid.
 21. A vaccine for treatment of a patient having a growth hormoneand/or insulin-like growth factor 1 deficiency, the vaccine comprising:an immunogenic amount of somatostatin-based antigen; and an adjuvantcomprising at least Carbopol 974P, squalene and arabinogalactan.
 22. Thevaccine of claim 21 wherein the somatostatin-based antigen issomatostatin-14 attached to an inactivated CAT enzyme.
 23. The vaccineof claim 22 wherein the inactivated CAT enzyme has one or more wild typehistidine residues replaced with alanine, glycine or other like aminoacid.